7 OCTOBER 2015

Scientific Background on the Nobel Prize in Chemistry 2015

M E C H A N I S T I C S T U D I E S O F D N A R E PA I R

compiled by the Class for Chemistry of the Royal Swedish Academy of Sciences

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The Royal Swedish Academy of Sciences has decided to award Tomas Lindahl, Paul Modrich

and Aziz Sancar the Nobel Prize in Chemistry 2015 for their “Mechanistic studies of DNA repair”

Damage to the genetic material poses a threat to all organisms. To counteract this threat, cells

have evolved a series of intricate DNA repair pathways that correct DNA lesions affecting base

pairing or structure of DNA. Today we understand the molecular mechanisms of these pathways

in great detail, in large part due to the pioneering studies by Lindahl, Modrich and Sancar that

opened up the field.

Background

The human genome encodes the information needed to create a complete human being. During

every cell division, more than three billion DNA base pairs are replicated and copies of the

genome are transferred to the daughter cells. Although very efficient, the DNA replication

machinery responsible for this task still makes occasional mistakes. Given the size of the human

genome and the large number of cells in a human body (about 3.7 × 1013) mistakes will

inevitably accumulate during the lifetime of an individual. Most of these errors will remain

silent, but they can also cause serious diseases.

Despite its essential role in storing genetic information, the DNA molecule has limited chemical

stability and is subject to spontaneous decay [1]. Processes such as hydrolysis and oxidation

occur at significant levels in vivo, in part due to reactive metabolites continuously generated in

various physiological processes. In addition, external factors like radiation and genotoxic

chemicals will further stimulate of DNA damage formation.

The inherent instability of DNA constitutes both an opportunity and a threat. DNA lesions can

block important cellular processes such as DNA replication and transcription, cause genome

instability and impair gene expression. Lesions can also be mutagenic and change the coding

capacity of the genome, which can lead to devastating diseases and conditions associated with

genome instability, including cancer, neurodegenerative disorders and biological ageing. At the

same time, without mutations Darwinian evolution would not be possible. Furthermore,

mutagenic chemicals and radiation can also have a healing effect; they can for instance be used

to treat cancer, by introducing DNA lesions that halt cell proliferation and stimulate

programmed cell death.

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The cell has developed ways to counteract DNA lesions and to keep DNA mutations at a

tolerable level. A number of different DNA repair mechanisms correct lesions and safeguard the

integrity of the genome. Four fundamental DNA repair pathways delineated by this year’s Nobel

Prize laureates will be discussed here.

Photoreactivation – the first DNA repair mechanism

In the 1920s, the American geneticist Hermann Muller (Nobel Prize in Physiology or Medicine,

1946) found that X-rays could mutate and kill cells [2]. Later on, other types of agents, including

ultraviolet (UV) light, were also shown to affect cell viability and mutation levels. The cellular

target for the lethal effects of X-rays and UV-light was unknown at this time, and there were no

identified cellular mechanisms that could repair the lesions once they occurred. A breakthrough

came in the late 1940s, when Albert Kelner studied bacteria and their recovery in response to

damage caused by UV-light. Kelner found that visible light could dramatically stimulate growth

recovery after a growth arrest caused by UV exposure [3], [4]. The phenomenon was termed

photoreactivation and it pointed to the existence of a light-dependent cellular mechanism that

could correct UV-induced cellular damage.

Oswald Avery and co-workers had demonstrated in 1944 that DNA is the material of heredity [5]

and in the 1950s it became clear that UV-induced damage most likely was targeted to DNA. At

this point, Renato Dulbecco (Nobel Prize in Physiology or Medicine, 1975) suggested that

photoreactivation was an enzymatic reaction dependent on visible light [6]. The correctness of

this assumption was demonstrated by Stanley Rupert, who in a series of reports showed that

DNA could be reactivated by visible light in the presence of a cell-free extract from Escherichia

coli or Saccharomyces cerevisiae [7, 8]. The observation of this enzymatic activity, known as the

photolyase, was of profound importance, since it demonstrated for the first time the existence of

DNA repair enzymes that could rescue UV-irradiated DNA. At first, the photolyase was just an

activity in an extract, but in 1978 Aziz Sancar could clone the E. coli photolyase gene and amplify

the gene product in vivo [9]. Sancar was at the time a PhD student of Rupert’s, but instead of

continuing to characterise the photolyase, he wrote his PhD dissertation and graduated. It

would take another six years, before Sancar returned to photolyase research.

Dark repair – the discovery of nucleotide excision repair

In addition to photoreactivation, UV damage can also be repaired in a light-independent process

(known as “dark repair”). That UV-irradiation of DNA introduced thymine dimers in vitro was

reported in 1960, but the in vivo relevance of this finding was unclear [10]. A couple of years

later, Jane Setlow and Richard Setlow demonstrated that thymine dimers inactivated

transforming DNA in the bacterium Hemophilus influenzae and that this type of lesion was

responsible for the biological effect of UV-radiation [11, 12]. This realization made it possible to

study the precise molecular consequences of thymine dimers and investigate how cells deal with

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them. In 1963, Richard Setlow reported that thymine dimers inhibit DNA synthesis and that

these lesions are excised from DNA in wild-type UV-irradiated bacteria, but not in an UV-

sensitive, mutant E. coli strain [13]. In 1964, he made the seminal discovery that thymine dimers

disappeared from the irradiated, high molecular weight genomic DNA shortly after exposure to

UV and instead appeared in low molecular weight fractions. Richard Setlow and his colleague

William Carrier correctly interpreted this result as thymine dimers being excised (removed)

from the DNA, hence the name excision repair [14]. Around this time, Richard Boyce and Paul

Howard-Flanders also published observations with conclusions similar to those reached by

Richard Setlow [15]. The emerging mechanisms of what later became known as nucleotide

excision repair (NER) was further elucidated in crucial work by Philip Hanawalt and David

Pettijohn, who found that UV-irradiation stimulated DNA repair synthesis even outside the S-

phase, i.e. independent of genome replication [16]. The groundbreaking contributions of these

early pioneers clearly indicated the existence of repair mechanisms that could correct UV-

induced lesions, but the precise molecular mechanisms underlying NER remained obscure.

The molecular mechanisms of NER

The identification of the enzymes responsible for NER was greatly assisted by genetics analyses.

Earlier bacterial work had identified uvrA, uvrB, and uvrC genes in a search for mutations that

impaired NER and hindered growth resumption after UV irradiation [17]. Work in vivo [18-20]

and in E. coli extracts [21] by Erling Seeberg and others indicated that the uvr gene products

functioned by endonucleolytic cleavage of irradiated DNA, but this could not be examined in

detail due to the lack of purified proteins.

In the 1970s, identification of proteins was a major challenge, since DNA sequencing techniques

were limited and a given gene locus could encode more than one protein. Aziz Sancar, now

working with W. Dean Rupp at Yale School of Medicine, developed the elegant Maxicell

technique, which relies on a UV-repair deficient bacterial strain [22]. After transformation of a

plasmid DNA of interest, the hypersensitive bacteria can be UV-irradiated, which causes

breakdown of the larger chromosomal DNA, whereas the plasmid molecules that have not been

hit by UV can continue to replicate and express proteins. In combination with radioactive amino

acid incorporation, the technique allows for labelling and detection of plasmid-encoded proteins

in the absence of a chromosomal background. The Maxicell technique was soon applied to a

wide variety of protein identification projects and allowed Sancar to rapidly identify the proteins

encoded by the uvrA, uvrB and uvrC genes [23-25].

In a groundbreaking work published in 1983 [26], Sancar used the purified UvrA, UvrB, and

UvrC proteins to reconstitute essential steps in the NER pathway. The three proteins acted

specifically on damaged DNA. With UV-irradiated DNA as a substrate, the proteins hydrolysed

two phosphodiester-bonds on the damaged DNA strand. The incisions were performed at

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precise locations relative the UV adduct, one at the 8th phosphodiester bond 5' to the lesion and

a second at the 4th or 5th bond 3' to the same lesion, thus generating a 12-13 nt long fragment.

Later, Sancar could show that the rate of the reaction is stimulated by UvrD (DNA helicase II)

and DNA polymerase I (Pol I), which catalyses the removal of the incised strand and synthesis of

the new DNA strand, respectively [27]. Finally, DNA ligase catalyses the formation of two new

phosphodiester bonds and thus seals the sugar-phosphate backbone. In the 1983 paper, Sancar

described the UvrA, B, and C proteins as working together in a single complex (an excinuclease),

but later findings by him and also others have modified this view. We know today that Uvr

proteins associate with DNA lesions in a step-wise fashion (Figure 1) [28]. First, a complex of

two UvrA and one UvrB subunit (UvrA2B) tracks along the DNA. The UvrA subunits are

responsible for the initial damage recognition on double-stranded DNA, which causes the

UvrA2B complex to halt. At this point, the UvrB helicase activity is activated, leading to local

unwinding of DNA around the lesion (about 5 bp), kinking of the template and further

recognition of the damaged strand by UvrB. As a consequence, the UvrA proteins dissociate

from UvrB and a single UvrC subunit binds to the remaining UvrB-DNA complex. UvrC

activates UvrB, which makes the 3' incision, which is followed by UvrC catalysed incision at the

5' side of the lesion. The UvrD helicase displaces the damaged DNA strand, after which only

UvrB remains bound to the gapped DNA. At this point, Pol I associates with DNA, fills the gap

and UvrB is released. Finally, the repaired patch is ligated.

We know today that thymine dimers are just one of numerous types of lesions that interfere with

normal base pairing and thus distort the helical structure of DNA. NER can recognise these

problems and correct them by its cut-and-patch mechanism. The overall mechanisms of NER in

mammalian cells are closely related to those characterised in bacteria. The mechanisms of

damage recognition, incision on both sides of the lesion, removal of the damaged oligomer and

resynthesis of the gap are very similar between the two systems. However, even if the overall

strategy of repair is the same, the proteins responsible are distinct. Whereas damage recognition

and dual incision is carried out by only three proteins in E. coli more than fifteen proteins are

required to carry out the same function in human cells.

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Mutations in the NER system is linked to a number of human genetic disorders, including

Xeroderma pigmentosum (XP), which is characterised by hypersensitivity to UV-radiation and

a very high risk for skin cancer. Many of the genes encoding mammalian NER factors were first

identified by James Cleaver, who carefully identified XP causing mutants in affected humans

[29]. Over the years, the mammalian NER system and its mechanisms have been characterised

in detail by a number of different laboratories. Among the many notable contributions is the

reconstitution of human NER with purified factors by Sancar [30] and Richard D. Wood [31].

The photolyase and its reaction mechanism

In 1982, Sancar took up a faculty position at the University of North Carolina, Chapel Hill. At

this point he also returned to his PhD project, the photolyase. Between 1984 and 1989, Sancar

published a series of papers in which he described the mechanisms for photolyase function,

including the identification of two chromophores present in the E. coli enzyme [32-34]. Sancar

demonstrated that photolyase, can convert the energy of an absorbed photon into chemistry that

Figure 1. (Petit and Sancar, Biochimie 1999, 81:15-25) A simplified model for nucleotide excision repair in E. coli. First, a heterotrimer of UvrA and UvrB is located to the DNA damage. Next, DNA is kinked and partially unwound by UvrB through an ATP-dependent reaction. UvrA leaves and UvrC binds to the UvrB-DNA complex, activating UvrB that makes the 3' incision, which is followed by an UvrC dependent 5' incision. The excised oligomer is released by the UvrD helicase. Pol I fills the gap and releases UvrB at the same time. Finally, the repaired patch is ligated.

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produces a localised free radical that initiates thymine dimer splitting. One of the two

chromophores in the photolyase is the fully reduced flavin–adenine dinucleotide (FADH-). This

chromofore functions as catalytic cofactor and upon excitation, it performs the actual repair,

when the excited flavin cofactor transfers an electron to the pyrimidine dimer to generate a

charge-separated radical pair (FADH· + Pyr<>Pyr·–). The anionic ring of the dimer is split by a

cycloreversion, and the excess electron returns to the flavin radical to restore the catalytically

competent FADH– form and close the catalytic photocycle (Figure 2). The reaction is light-

activated, since the flavin cofactor may be excited by direct photon absorption or by resonance

energy transfer from another chromophore, which is an antenna pigment

(methenyltetrahydrofolate or deazaflavin) that harvests sunlight and enhances repair efficiency.

Photoreactivation was the first form of DNA repair identified, but this process is not conserved

in mammalian cells, which instead rely on NER for correction of UV damage. However, the

photolyase has mammalian homologues, which are used to help setting the circadian clock, i.e.

the regulation of biological processes in response to light [35].

DNA is an unstable molecule

In the early 1970s Tomas Lindahl demonstrated that DNA has limited chemical stability even in

the absence of external physical assaults. Under physiological conditions DNA is subject to a

number of chemical reactions such as hydrolytic deamination, oxidation and non-enzymatic

methylation. These reactions modify the bases of DNA and as a consequence increase the risk

for mutations. Tomas Lindahl used the term DNA decay to describe these processes and

elegantly demonstrated that under physiological conditions, spontaneous hydrolytic DNA

Figure 2. (Cao et al., Proc Natl Acad Sci USA 2005, 102: 16128-32) Repair of damaged DNA by photolyase. Photolyase acts through an electron-transfer radical mechanism. The key catalytic reactions, charge separation (k 1) and ring splitting (k 2), are given at the bottom.

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depurination occur at significant levels [36] and stimulate cleavage of DNA chains [37]. Perhaps

the most fascinating discovery was the demonstration of high levels of spontaneous cytosine

deamination under physiological conditions, which leads to the formation of uracil. Since uracil

forms base pairs with adenine, cytosine deaminiation is a highly mutagenic process, with

important long-term consequences. High levels of cytosine deamination pose a risk of depleting the

genetic material from cytosine-guanine base pairs and replacing them with thymine-adenine [38].

Discovery of base excision repair

Based on his observation that uracil is frequently formed in DNA, Tomas Lindahl came to the

conclusion that there must exist an enzymatic pathway that can handle this and other types of

base lesions. In a now classic study, he single-handedly identified the E. coli uracil-DNA

glycosylase (UNG) as the first repair protein [39] and two years later a second glycosylase,

specific for 3-methyladenine DNA [40]. We know today that UNG is the founding member of a

large family of proteins that orchestrate base excision repair (BER). The identification of UNG

relied on careful analysis of enzymatic release of uracil as a free base from DNA in vitro. Lindahl

demonstrated that the enzyme was specific to DNA and did not act on deoxymononucleotides or

any form of RNA. He also showed that the DNA backbone remained intact in the process, which

immediately suggested the involvement of another category of enzymes, the

apurinc/apyrimidinic endonucleases. An E. coli activity specific for apurinic sites had been

identified just two years earlier by Walter Verly [41, 42]. Lindahl could outline the basic

concepts for BER already in the 1974 paper and in a series of papers published in the years to

follow he and others could verify the proposed model. Through continued studies, Lindahl could

reconstitute the entire BER with purified enzyme, both from E. coli [43] and human cells [44].

The process is initiated when a DNA glycosylase recognises and hydrolytically cleaves the base-

deoxyribose glycosyl bond of a damaged nucleotide (Figure 3). Mammalian cells contain

number of different DNA glycosylases, which act on various forms of base modifications [45].

Once a damaged nucleotide has been identified, the DNA glycosylase kinks the DNA and the

abnormal nucleotide flips out [46]. The altered base interacts with a specific recognition pocket

in the glycosylase and is released by cleavage of the glycosyl bond. The DNA glycosylase itself

often remains bound to the abasic site until being replaced by the next enzyme in the reaction

cycle, the apurinic/apyrimidinic (AP) endonuclease, which cleaves the DNA backbone at the 5′

side of the abasic position. The AP endonuclease also associates with DNA polymerase β (pol-β),

to fill the gap [47]. In addition, pol-β harbors a lyase activity, which excises the 5′-terminal base-

free sugar phosphate residue in a nonhydrolytic elimination process [48]. However, the repair of

oxidatively damaged nucleotides has no requirement for such a pol-β-associated lyase activity,

because the DNA glycosylases concerned possess endogenous AP lyase activity themselves. In a

final step, DNA ligase III/XRCC1 heterodimer interacts with pol-β, displaces the polymerase,

and catalyses the formation of a new phosphodiester bond [44].

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We know today that BER corrects many different forms of lesions that affect the bases without

causing gross structural perturbation in the DNA structure. Lesions like these present a special

challenge to the replication machinery, since they may cause miscoding, i.e. the chemically

altered base has the potential to base pair with an erroneous base, either during DNA replication

or transcription. To date, more than 100 different types of oxidative lesions have been identified

and the vast majority of these are corrected by BER. The lesions corrected by BER may also be

formed by exogenous factors, including ionizing radiation.

Figure 3. (Lindahl, Nature 1993, 362:709-715) The base excision repair pathway for removal of endogenous damage from cellular DNA. The scheme shows the removal of a deaminated cytosine residue (uracil). The details of the reaction are given in the text.

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Mismatch repair

As noted above, the DNA replication machinery is not error-free. There is always the possibility

that an incorrect nucleotide is introduced during synthesis of a new DNA strand. As a result, a

non-Watson-Crick base pair is formed, which distorts the double-stranded DNA helix. These

types of errors are known as mismatches and they have the capacity to change the sequence of

DNA, i.e. to introduce mutations. As a first line of defence against mismatches, replicating DNA

polymerases contain a 3′ to 5′ exonuclease activity that allows them to proofread the newly

synthesized DNA strand [49]. The exonuclease activity can correct mistakes during DNA

replication by reversing the direction of the polymerase and excising incorrectly introduced

nucleotides. Even if proofreading efficiently corrects most mistakes made during DNA synthesis,

some non-Watson-Crick base pairs still remain. To correct these errors, cells use mismatch

repair. It is estimated that that replicative DNA polymerases with proofreading in vitro have

error frequencies of about 5 × 10-5. Mismatch repair lowers this frequency significantly and the

mutation rate in for example human germ cells is estimated to be close to 1 × 10-8 per base pair

per generation [50].

The early studies of mismatch repair were mainly carried out by geneticists interested in

recombination. Robin Holliday had hypothesised that heteroduplexes between different DNA

strands would be formed during recombination [51]. The mismatches formed in this way had to

be corrected by some sort of cellular system, leading to gene conversion. Indeed, transfection of

heteroduplex molecules of lambda phage DNA into E. coli led to repair of the mismatches, but

the enzymatic system required for this effect was unknown. An important step was taken in

1976, when Robert Wagner and Matthew Meselson reported that repair of two or more close

sites on the same heteroduplex DNA molecule occur more often on the same strand than on the

opposite strand [52]. This observation led them to propose that mismatches are repaired by a

strand-specific mechanism directed towards one strand, which allows for repair of long DNA

tracts. They also speculated that strand-specific mismatch repair could be used to correct non-

canonical base pairs formed during DNA synthesis. The repair machinery could be guided to the

newly replicated strand either via a special relation to the replication machinery or directed by

under-methylation of the newly synthesised strand. In E. coli DNA is normally methylated on

both strands at GATC-sites, but during DNA synthesis the nascent strand is unmethylated for a

period of time [53], which could allow the mismatch repair machinery to distinguish newly

replicated DNA from the template DNA strand. In support of this notion, loss of cellular DNA

methylase, the enzyme that adds a methyl group to the adenine of the sequence GATC, was

already in 1975 shown to cause higher mutation rate in E. coli [54]. The genetics of mismatch

repair was further clarified when Barry W. Glickman and Miroslav Radman could demonstrate

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that mutH, mutL, mutS and uvrD genes were all required for the methylation-instructed DNA

mismatch correction [55].

Direct evidence for methyl-directed repair of mismatches came in 1983. First, Paul Modrich and

Matthew Meselson used heteroduplex constructs with defined states of DNA methylation to

demonstrate that DNA methylation indeed directed strand-specific elimination of mismatches

in E. coli [56]. Furthermore, Modrich developed an assay that allowed analysis of DNA

mismatch repair in cell-free E. coli extracts [57]. In his assay, he used the classical bacteriophage

heteroduplexes, but introduced mismatches within overlapping sequences recognised by two

different restriction endonucleases. About 1,000 bp from the mismatch was a GATC methylation

site, which could be methylated in a controlled way and thus used to monitor strand-specific

correction. Using the assay, Modrich could demonstrate that the repair activity was dependent

on ATP, the methylation state of the heteroduplex, and that mutations affecting mutH, mutL,

mutS, and uvrD all impaired mismatch repair in cell-free E. coli extracts. With the help of this

elegant assay, Modrich could in a series of papers isolate the products of the different repair

genes to near homogeneity, identify the proteins and investigate their properties in vitro in

great detail [58-60].

Modrich’s work culminated in a groundbreaking paper published in 1989 [61], in which he could

finally reconstitute DNA mismatch correction in a defined in vitro system. In the paper,

Modrich demonstrated the requirement of DNA polymerase III, exonuclease I, and DNA ligase

for mismatch repair. He then combined these factors with purified MutH, MutL, MutS, UvrD,

and single-stranded DNA-binding protein. Together these factors could process mismatches in

vivo in a strand-specific manner directed by the single, GATC sequence methylated on only one

strand (hemimethylated) and located distant from the mismatch. In this and other studies,

Modrich demonstrated that MutS, which recognises and binds to non-Watson-Crick base pairs,

performs a core function in mismatch repair system. To confer strand-specificity, MutH binds at

hemimethylated GATC sites on the nascent strand. MutL acts as a mediator, which interacts

with both MutH and MutS. MutL transduces signals from MutS, which leads to activation of the

latent MutH endonuclease activity causing a nick in the nascent DNA strand near the

hemimethylated GATC-site. The machinery now interacts with a helicase (UvrD), which

together with the MutS, MutL, and MutH proteins separates the two DNA strands towards the

location of the mismatch. Displacement of the mutant strand continues past, and halts just

downstream of, the mismatch. The nascent strand is then replaced by a gap-filling reaction, in

which DNA polymerase III uses the parental strand as a template.

Mismatch repair in mammalian cells.

Later studies by Modrich and others have demonstrated the conservation of mismatch repair in

eukaryotic cells and in 2004, Modrich managed to reconstitute human mismatch repair with

only purified factors [62]. In contrast to the situation in E. coli, DNA methylation does not direct

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strand specific DNA repair in eukaryotic cells. One possibility is that the strand specific nicks

formed during DNA replication can direct strand-specific error correction. In support of this

notion, mismatch repair is more efficient on the lagging strand at the replication fork [63], and a

single nick is sufficient to direct strand specific repair in in vitro [62, 64]. Alternatively, the

mismatch repair machinery may be directed by ribonucleotides transiently present in DNA after

replication [65]. In other aspects, eukaryotic mismatch repair is closely related to the E. coli

system with conserved homologues to key factors such as MutS and MutL. The importance of

the mammalian mismatch repair system is underscored by the finding that defects in this

pathway cause hereditary nonpolyposis colon cancer [66, 67].

Summary

Tomas Lindahl, Paul Modrich, and Aziz Sancar have made fundamental and groundbreaking

discoveries on the enzymatic mechanisms of DNA repair. Lindahl demonstrated that DNA is an

inherently unstable molecule, subject to decay even under physiological conditions. Guided by

thia observation, Lindahl identified a completely new group of DNA glycosylases and described

their role in base excision repair. Modrich transformed the field of mismatch repair from genetic

observations to a detailed biochemical understanding, first in bacteria, and later in eukaryotic

cells. Sancar has transformed the field of nucleotide excision repair, from genetics and

phenomena in cell extracts, to a detailed molecular description of the mechanisms involved, first

in bacteria, and later also in eukaryotic cells. Sancar also explained the molecular mechanisms

underlying photoreactivation, the first form of DNA repair described.

Claes M. Gustafsson

Professor of Medical Chemistry and Cell Biology, University of Gothenburg

Member of the Nobel Committee for Chemistry

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